Compounds capable of binding specifically to a target single-stranded genetic sequence have many diagnostic and therapeutic applications. For example, in diagnostic applications, the compounds can be used to "hybridize" with analyte single-stranded polynucleotides, to detect with high sensitivity the presence of specific nucleic acid species characteristic of a pathogenic state. In therapeutic applications, the compounds can bind to and inactivate viral genomes, or other single-stranded polynucleotide species characteristic of a pathogenic state. In principle most or all viral diseases and many other diseases may be treatable with suitable sequence-specific gene inactivating agents capable of selectively blocking or reducing the expression in vivo of the offending genes or gene transcripts.
Compounds which are designed to block targeted genetic sequences within living cells can be divided into two general classes:
One class of compounds include sequence-specific nucleic acids, and derivatives thereof, having charged phosphodiester internucleoside linkages. These compounds typically are anti-sense polynucleotide stands designed to bind to and inactivate a target "sense" strand. In spite of the promising work on delivery of modified oligonucleotides into cells in culture (Lamaitre), oligonucleotides coding for or constituting anti-sense nucleic acid sequences probably have little near-term potential for treating viral diseases because of the difficulty in introducing intact nucleic acids into target tissues of the body, principally because nucleic acids (and especially high molecular weight complexes thereof) are rapidly sequestered by the reticuloendothelial system. Even when the nucleic acids are incorporated into liposomes or related delivery vehicles, evasion of the reticuloendothelial cells presents a difficult challenge.
A second class of compounds designed for binding to and blocking target genetic sequences includes nucleic acid analogs with substantially uncharged backbones. Such analogs have the potential for: a) an enhanced rate of passage into living cells (presumably by phase transfer across cell membranes); b) resistance to intracellular enzymatic degradation; and, c) defeating normal cellular mechanisms for strand separating the analog/target duplex. Pioneering work along this line was carried out in the early 1970's (Pitha, 1970b). These workers prepared a variety of homopolymeric polynucleotide analogs in which the normal sugar-phosphate backbone of nucleic acids was replaced by a polyvinyl backbone. The nucleic acid analogs were reported to have the expected Watson/Crick pairing specificities with complementary polynucleotides, but with substantially reduced Tm values (Pitha, 1970a). Subsequently, a variety of other uncharged polynucleotide analogs were reported (Jones, 1967, 1973; Gait; Cassidy; Buttrey).
There are, however, a variety of problems inherent in the structures of uncharged polynucleotide analogs of the type mentioned above. The structures are unstable in aqueous solution; do not allow assembly of different subunits in a defined order; and/or, the base-pairing moieties are not properly spaced for efficient binding to a target sequence. Further, molecular modeling studies carried out in support of the present invention indicate that in some structures the base-pairing moieties are linked too closely to the backbone to allow effective binding of the base-pairing moieties to contiguous bases of a complementary polynucleotide, while in other structures, there are excessive degrees of freedom for the base-pairing moieties, permitting undesired pairing of the moieties with noncomplementary (in the Watson/Crick sense) bases of a polynucleotide.
More recently, uncharged polynucleotide analogs which are nearly isostructural with nucleic acids have been reported. These substances contain chiral intersubunit linkages and include both aryl nitrogen mustard-derivatized (Karpova) and underivatized (Tullis) oligonucleotides having their phosphates in the uncharged triester state. In addition, polynucleotide analogs containing uncharged methylphosphonate linkages between the deoxyribonucleoside subunits have been prepared. These uncharged oligonucleotide analogs have been reported to enter living cells and pair with complementary genetic sequences therein. The methylphosphonate-linked analogs have the further merit of resistance to degradation by nucleases (the phosphotriester-linked analogs appear to be subject to conversion to diesters by an esterase, with subsequent cleavage by nucleases) (Miller, 1979, 1980; Jayaraman; Murakami; Blake, 1985a, 1985b; Smith). 0f particular note, the methylphosphonate-linked oligonucleotide analogs have been used successfully to specifically block globin synthesis in rabbit reticulocytes, protein synthesis (N, NS, and G proteins) of Vesicular stomatitis virus in VSV-infected mouse L-cells; T-antigen synthesis in SV40-infected African green monkey kidney cells; and, reproduction of Herpes simplex virus in HSV-infected Vero and human foreskin fibroblast cells.
Despite the potential advantages of uncharged, relatively isostructural oligonucleotide analogs discussed above, the compounds are limited in practical application due to the chiral backbone centers at each methyl phosphonate linkage. Because the methyl moiety on the phosphonate of the internucleoside linkage generates a chiral center, the compounds have atactic (i.e., a random sequence of chiral centers) backbones. Such stereoirregular backbones result in a broad range of analog/target binding constants--the mean of which is substantially reduced in comparison to the binding constant expected for a corresponding stereoregular polynucleotide analog and its complementary polynucleotide. For the methylphosphonate-linked nucleic acid analogs this binding constant reduction and broadening derives from one linkage isomer favoring Watson/Crick pairing of proximal bases and the other linkage isomer inhibiting Watson/Crick pairing of proximal bases. This linkage chirality effect on base-pairing is illustrated in a report which characterizes the two stereo-isomeric forms of a methylphosphonate-linked di(deoxyribonucleoside) (Miller, 1979). The isomeric dimers were coupled via phosphodiester linkages to give partially charged homoisomeric decamers. One such decamer containing the preferred isomeric methylphosphonate linkages paired with its complementary DNA sequence with a Tm of 33.degree. C. In contrast, the corresponding decamer, containing the other isomeric form of methylphosphonate linkages, had a Tm value approximately 30.degree. C. lower (Miller)--suggesting that each linkage having the suboptimal chirality reduces the Tm by about 6.degree. C. Thus, for a methylphosphonate-linked 15-mer, one would predict a given preparation, when paired with its target genetic sequence, would exhibit Tm values over an 84.degree. C. range, with a mean Tm approximately 42.degree. C. lower than for the case of a corresponding specie having all linkages of the preferred chirality.
One feature then of the above-described compounds is that a given preparation will contain multiple molecular species, each with a different target binding constant. Those species having lower binding constants will contribute little or nothing to the target binding activity, while some of those having significantly higher binding constants may also form moderately stable mispaired complexes with nontarget sequences. In therapeutic use, the presence of weak-binding and nonbinding components in the preparation can increase the dosage required to achieve a desired therapeutic effect--possibly by as much as two or three orders of magnitude. Further, any significant binding to nontarget sequences by the strongest-binding components increases the possibility of toxic side effects due to inactivation of inherent sequences in the patient.
We have previously described a class of polynucleotide binding polymers having intersubunit linkages which are both uncharged and achiral. U.S. patent applications Ser. No. 712,396, filed 15 Mar. 1985, abandoned; Ser. No. 911,258, filed 24 Sep. 1986, abandoned; Ser. No. 944,707, filed 18 Dec. 1986; and PCT patent application Ser. No. US86/00544, filed 14 Mar. 1986. The PCT application is incorporated by reference into the present application. Molecular modeling studies of the compounds, which include carbonate-linked deoxyribonucleosides and carbamate-linked 5'-amino-2',5'-dideoxyribonucleosides, indicate that the component bases in the polymers are properly positioned and spaced for Watson/Crick pairing to complementary genetic sequences. An important advantage of these compounds is that the backbone linkages are achiral, as in natural polynucleotides, and therefore the molecules of the preparation will have uniform binding constants to target single-strand polynucleotides.
In the course of further molecular modeling work, combined with synthesis and target binding studies, several polymers originally described in the above cited patent applications have been identified as preferred structures, either because of better stability, greater ease of synthesis, or improved binding. The present application describes the preferred structures, related polymer structures and synthetic routes for producing the structures.